CN112795636A - Application of reagent for detecting miR-122 in liver tissue in preparation of liver cirrhosis screening kit - Google Patents
Application of reagent for detecting miR-122 in liver tissue in preparation of liver cirrhosis screening kit Download PDFInfo
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- 208000019425 cirrhosis of liver Diseases 0.000 title claims abstract description 57
- 108091007780 MiR-122 Proteins 0.000 title claims abstract description 41
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 39
- 108091051828 miR-122 stem-loop Proteins 0.000 title claims abstract description 38
- 210000005228 liver tissue Anatomy 0.000 title claims abstract description 32
- 238000012216 screening Methods 0.000 title claims abstract description 27
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 230000007882 cirrhosis Effects 0.000 claims abstract description 38
- 206010016654 Fibrosis Diseases 0.000 claims abstract description 37
- 208000002672 hepatitis B Diseases 0.000 claims abstract description 23
- 239000012807 PCR reagent Substances 0.000 claims description 18
- 238000001514 detection method Methods 0.000 claims description 18
- 108020004707 nucleic acids Proteins 0.000 claims description 12
- 150000007523 nucleic acids Chemical class 0.000 claims description 12
- 102000039446 nucleic acids Human genes 0.000 claims description 12
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 238000003753 real-time PCR Methods 0.000 claims description 8
- 238000012408 PCR amplification Methods 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 4
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 4
- 238000012163 sequencing technique Methods 0.000 claims description 4
- 230000003321 amplification Effects 0.000 claims 2
- 238000003199 nucleic acid amplification method Methods 0.000 claims 2
- 241000700721 Hepatitis B virus Species 0.000 description 8
- 108700011259 MicroRNAs Proteins 0.000 description 7
- 239000002679 microRNA Substances 0.000 description 7
- 101000884191 Homo sapiens Cyclin-G1 Proteins 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 208000015181 infectious disease Diseases 0.000 description 6
- 102100038252 Cyclin-G1 Human genes 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 239000005089 Luciferase Substances 0.000 description 4
- 201000007270 liver cancer Diseases 0.000 description 4
- 208000014018 liver neoplasm Diseases 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 108091069016 Homo sapiens miR-122 stem-loop Proteins 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 241000700605 Viruses Species 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000011901 isothermal amplification Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
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- 208000000419 Chronic Hepatitis B Diseases 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 108090000331 Firefly luciferases Proteins 0.000 description 1
- 206010019663 Hepatic failure Diseases 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 108010052090 Renilla Luciferases Proteins 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 238000012226 gene silencing method Methods 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 231100000835 liver failure Toxicity 0.000 description 1
- 208000007903 liver failure Diseases 0.000 description 1
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 238000002616 plasmapheresis Methods 0.000 description 1
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- 238000002604 ultrasonography Methods 0.000 description 1
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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Abstract
The invention provides application of a reagent for detecting miR-122 in liver tissues in preparation of a cirrhosis screening kit. The invention discovers for the first time that the expression level of miR-122 in liver tissues of hepatitis B cirrhosis patients is obviously lower than that of liver tissues of healthy people (P < 0.01). Therefore, the reagent for detecting miR-122 in liver tissues can be used for preparing a hepatitis B cirrhosis screening kit, and effective screening of hepatitis B cirrhosis is realized. The screening kit provided by the invention can quickly, efficiently and accurately screen and diagnose patients with liver cirrhosis (particularly hepatitis B cirrhosis), and provides a new choice for the liver cirrhosis screening kit.
Description
Technical Field
The invention belongs to the field of in-vitro diagnostic reagents, and particularly relates to application of a reagent for detecting miR-122 in liver tissues in preparation of a cirrhosis screening kit.
Background
Hepatitis B Virus (HBV) infection is prevalent worldwide, and it has been reported by the world health organization that about 20 million people worldwide have been infected with HBV, of which 2.4 million people are chronic HBV infected and about 65 million people die each year from liver failure, cirrhosis and hepatocellular carcinoma due to HBV infection. In patients with global liver cirrhosis, the proportion caused by HBV infection is 30%, and in patients with liver cirrhosis in our country, the proportion caused by HBV infection is 60%.
The current clinical diagnosis means of cirrhosis mainly comprise color Doppler ultrasound, CT, gastroscope, plasmapheresis indexes and the like. However, the clinical application of these single techniques or indexes has some limitations and disadvantages, and none of them can accurately and timely diagnose the progress of liver cirrhosis. Simple and efficient diagnosis methods for liver cirrhosis caused by HBV infection (hepatitis B cirrhosis for short) are lacked.
microRNA (miRNA) is a non-coding single-stranded small RNA molecule, can realize gene silencing after transcription by degrading target genes or inhibiting the translation of the target genes, and plays an important role in the generation and development of diseases through the regulation and control of downstream target genes. Recent studies have shown that cirrhosis associated with HBV infection is closely related to miRNA, which can affect the aforementioned disease progression by acting on the virus itself or on the immune system. Research shows that the miRNA expression profile of a liver disease patient infected by virus is obviously different from the miRNA expression profile of healthy human tissues. Researchers also find that a large amount of stable small ribonucleic acid molecules, namely miRNA exist in human serum/plasma, which lays a foundation for clinically diagnosing liver cirrhosis by detecting the expression quantity of the microRNA molecules in the serum/plasma.
Chinese patent application No. CN2009100514930 discloses a kit for early prediction of the development of chronic hepatitis B into cirrhosis by detecting the content of hsa-mir-122 in plasma or serum. The application discloses that the plasma of patients with cirrhosis has a higher hsa-mir-122 content than that of healthy people, which is about 3 times higher than that of healthy people. However, this method is to perform early prediction of liver cirrhosis by detecting the hsa-mir-122 content in plasma or serum, which may be affected by tissues other than liver, affecting the detection accuracy.
At present, no report is found for screening liver cirrhosis by detecting miR-122 levels in liver tissues.
Disclosure of Invention
The invention aims to provide application of a reagent for detecting miR-122 in liver tissues in preparation of a cirrhosis screening kit.
miRNA-122, abbreviated as miR-122.
The invention provides application of a reagent for detecting miR-122 in liver tissues in preparation of a cirrhosis screening kit.
Further, the liver cirrhosis is hepatitis b liver cirrhosis.
Further, the reagent is a PCR reagent, a nucleic acid isothermal amplification reaction reagent, a nucleic acid blotting detection reagent, a colloidal gold detection reagent, a nucleic acid sequencing reagent or a gene chip detection reagent.
Further, the PCR reagent is a fluorescent quantitative PCR reagent.
Further, the PCR reagent comprises a primer for carrying out PCR amplification on miR-122, and the sequence of the primer is shown as SEQ ID NO.1 and SEQ ID NO. 2.
The invention also provides a cirrhosis screening kit, which comprises a reagent for detecting miR-122 in liver tissues.
Further, the reagent is a PCR reagent, a nucleic acid isothermal amplification reaction reagent, a nucleic acid blotting detection reagent, a colloidal gold detection reagent, a nucleic acid sequencing reagent or a gene chip detection reagent.
Further, the PCR reagent is a fluorescent quantitative PCR reagent.
Further, the PCR reagent comprises a primer for carrying out PCR amplification on miR-122, and the sequence of the primer is shown as SEQ ID NO.1 and SEQ ID NO. 2.
The invention discovers for the first time that the expression level of miR-122 in liver tissues of hepatitis B cirrhosis patients is obviously lower than that of liver tissues of healthy people (P < 0.01). Therefore, the reagent for detecting miR-122 in liver tissues can be used for preparing a hepatitis B cirrhosis screening kit, and effective screening of hepatitis B cirrhosis is realized. The screening kit provided by the invention can quickly, efficiently and accurately screen and diagnose patients with liver cirrhosis (particularly hepatitis B cirrhosis), and provides a new choice for the liver cirrhosis screening kit.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Detailed Description
The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.
Example 1: composition of hepatitis B cirrhosis detection kit
The hepatitis B cirrhosis detection kit provided by the invention consists of a fluorescent quantitative PCR reagent for detecting miR-122 in human liver tissues. The fluorescent quantitative PCR reagent comprises a primer for carrying out PCR amplification on miR-122, and the sequences of the primer are shown in SEQ ID NO.1 and SEQ ID NO. 2:
5′-GTCAGATCTATGCCTGTAATCCCAGCACTT-3′(SEQ ID NO.1)；
5′-GTCAAGCTTTCTTTTTCAACACCGGAGTCA-3′(SEQ ID NO.2)。
example 2: expression test of miR-122 in liver tissue of hepatitis B cirrhosis patients
1. Experimental methods
Real-time fluorescent quantitative PCR (qRT-PCR) is adopted to detect the miR-122 levels in liver tissues of healthy people, liver tissues of hepatitis B cirrhosis patients and liver tissues of liver cancer patients respectively. The specific method comprises the following steps:
(1) clinical data
20 patients with hepatitis B and cirrhosis, 20 patients with liver cancer and 20 healthy controls were selected.
(2) Detection method
Total RNA of liver tissue cells of hepatitis B cirrhosis patients, liver cancer patients and healthy controls is respectively extracted by adopting an RNA extraction kit (Tiangen Biochemical technology (Beijing) Co., Ltd.) and is reversely transcribed to obtain cDNA. And performing PCR amplification on the miR-122 by using a quantitative PCR instrument (Bio-RAD), and quantitatively analyzing the expression level of the miR-122 by using a 2-delta Ct method. miR-122 upstream primer sequence: 5'-GTCAGATCTATGCCTGTAATCCCAGCACTT-3' (SEQ ID NO.1), and a downstream primer sequence 5'-GTCAAGCTTTCTTTTTCAACACCGGAGTCA-3' (SEQ ID NO. 2).
Processing was performed using SPSS version 20.0 software. Data are expressed as mean ± standard deviation, two groups are tested by t test, and the comparison between groups is analyzed by one-way variance. Differences with P <0.05 were statistically significant.
2. Results of the experiment
TABLE 1 results of miR-122 expression level determination in liver tissues of different populations
| Group of | Relative expression levels of miR-122 |
| Normal liver tissue | 3.56±0.54 |
| Hepatitis B cirrhosis tissue | 1.15±0.23* |
| Liver cancer tissue | 0.17±0.04* |
In table 1, P <0.01 compared to normal liver tissue.
The results show that the expression level of miR-122 in the liver tissue of the hepatitis B cirrhosis patients is significantly lower than that of the liver tissue of healthy people (P < 0.01).
Example 3: verification of miR-122 target gene CCNG1 by using dual-luciferase experiment
(1) Experimental methods
A dual-luciferase experiment is adopted to verify the target regulation effect of miR-122 on CCNG 1.
According to 3x104HEK293T cells are inoculated in a 96-well plate, miR-122mimics or NC is transfected after 24 hours, or a plasmid containing miR-122 combined with a CCNG 13 'UTR sequence or NC of a CCNG 13' UTR sequence is transfected after 48 hours, the activities of firefly luciferase and renilla luciferase are determined through a dual-luciferase reporter gene detection system, 3 parallel wells are arranged in each group and repeated for 3 times, the ratio is calculated and compared with the difference of each group, and the influence of miR-122 on CCNG1 expression is observed. The results are shown in Table 2.
(2) Results of the experiment
TABLE 2 Dual luciferase assay results
In table 2, P is <0.01 compared to Mimic NC.
The double-luciferase activity detection result shows that the target gene of miR-122 is CCNG 1.
In conclusion, the invention provides application of a reagent for detecting miR-122 in liver tissues in preparation of a cirrhosis screening kit. The invention discovers for the first time that the expression level of miR-122 in liver tissues of hepatitis B cirrhosis patients is obviously lower than that of liver tissues of healthy people (P < 0.01). Therefore, the reagent for detecting miR-122 in liver tissues can be used for preparing a hepatitis B cirrhosis screening kit, and effective screening of hepatitis B cirrhosis is realized. The screening kit provided by the invention can quickly, efficiently and accurately screen and diagnose patients with liver cirrhosis (particularly hepatitis B cirrhosis), and provides a new choice for the liver cirrhosis screening kit.
SEQUENCE LISTING
<110> institute of medical accomplishment
Application of reagent for detecting miR-122 in liver tissue in preparation of liver cirrhosis screening kit
<130> GYKH1825-2020P0112290CCR4
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 30
<212> DNA
<213> Artificial sequence
<400> 1
gtcagatcta tgcctgtaat cccagcactt 30
<210> 2
<211> 30
<212> DNA
<213> Artificial sequence
<400> 2
gtcaagcttt ctttttcaac accggagtca 30
Claims (9)
1. Application of a reagent for detecting miR-122 in liver tissues in preparation of a cirrhosis screening kit.
2. Use according to claim 1, characterized in that: the liver cirrhosis is hepatitis B cirrhosis.
3. Use according to claim 1 or 2, characterized in that: the reagent is PCR reagent, nucleic acid constant temperature amplification reaction reagent, nucleic acid blotting detection reagent, colloidal gold detection reagent, nucleic acid sequencing reagent or gene chip detection reagent.
4. Use according to claim 3, characterized in that: the PCR reagent is a fluorescent quantitative PCR reagent.
5. Use according to claim 3 or 4, characterized in that: the PCR reagent comprises a primer for carrying out PCR amplification on miR-122, and the sequence of the primer is shown as SEQ ID NO.1 and SEQ ID NO. 2.
6. A cirrhosis screening kit, which is characterized in that: it includes reagents for detecting miR-122 in liver tissue.
7. The cirrhosis screening kit of claim 6, wherein: the reagent is PCR reagent, nucleic acid constant temperature amplification reaction reagent, nucleic acid blotting detection reagent, colloidal gold detection reagent, nucleic acid sequencing reagent or gene chip detection reagent.
8. The cirrhosis screening kit of claim 7, wherein: the PCR reagent is a fluorescent quantitative PCR reagent.
9. The cirrhosis screening kit of claim 7 or 8, characterized in that: the PCR reagent comprises a primer for carrying out PCR amplification on miR-122, and the sequence of the primer is shown as SEQ ID NO.1 and SEQ ID NO. 2.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101368213A (en) * | 2008-10-13 | 2009-02-18 | 南京大学 | Blood serum minuteness ribonucleic acid reagent kit and application in early diagnosis of hepatitis B |
| CN101555520A (en) * | 2009-05-19 | 2009-10-14 | 中国人民解放军第二军医大学 | Has-mir-122 kit for early prediction of hepatocirrhosis developed from chronic hepatitis B and detection method thereof |
| CN102206702A (en) * | 2010-03-29 | 2011-10-05 | 北京大学 | miR-122 (micro-ribonucleic acid-122) as serum marker for liver diseases |
| CN106337051A (en) * | 2015-07-09 | 2017-01-18 | 首都医科大学附属北京佑安医院 | Hepatitis b/hepatic cirrhosis microRNA molecular marker combination and purpose thereof |
| CN111826434A (en) * | 2019-04-19 | 2020-10-27 | 北京旷博生物技术股份有限公司 | Application of microRNA marker miR-122-5p in hepatic fibrosis diagnosis |
-
2021
- 2021-01-07 CN CN202110019250.XA patent/CN112795636A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101368213A (en) * | 2008-10-13 | 2009-02-18 | 南京大学 | Blood serum minuteness ribonucleic acid reagent kit and application in early diagnosis of hepatitis B |
| CN101555520A (en) * | 2009-05-19 | 2009-10-14 | 中国人民解放军第二军医大学 | Has-mir-122 kit for early prediction of hepatocirrhosis developed from chronic hepatitis B and detection method thereof |
| CN102206702A (en) * | 2010-03-29 | 2011-10-05 | 北京大学 | miR-122 (micro-ribonucleic acid-122) as serum marker for liver diseases |
| CN106337051A (en) * | 2015-07-09 | 2017-01-18 | 首都医科大学附属北京佑安医院 | Hepatitis b/hepatic cirrhosis microRNA molecular marker combination and purpose thereof |
| CN111826434A (en) * | 2019-04-19 | 2020-10-27 | 北京旷博生物技术股份有限公司 | Application of microRNA marker miR-122-5p in hepatic fibrosis diagnosis |
Non-Patent Citations (1)
| Title |
|---|
| 孙艳等: "肝特异性miR-122启动子的克隆与组织特异性研究", 《中国科技论文在线》 * |
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